Summary of Work: This project includes those endeavors in which the mass spectrometry workgroup collaborates with other groups, both inside and outside the Institute to solve problems of mutual interest. The major focuses of these projects are: 1) structure determination of unknown compounds; 2) identification and/or confirmation of biological pathways; 3) quantitation; and 4) development of strategies for the analysis of biologically important compounds. Not included in this project are the many analyses performed on a strictly service basis. We continue to collaborate with D. Zeldin, LPP, on the identification and quantitation of arachidonic acid metabolites in physiological samples by selected ion monitoring (SIM) under negative ion chemical ionization (NICI) mass spectrometry. The products of enzymatically induced arachidonic acid oxidation, epoxyeicosatrienoic acids (EETs) and hydroxyeicosatrienoic acids (HETEs), exhibit significant biological effects, both harmful and beneficial. The characterization of the enzymes involved in the oxidation process and location of the enzymes is important in understanding the functional significance of the enzymes. Additionally, the response of the enzymes involved with arachidonic acid metabolism to environmental challenges may be a significant factor/indicator of environmentally induced disorders. In addition to the arachidonic acid metablolites, we are quantitating isoprostanes in the urine of male and female CYP2J5 knockout mice and control wild-type mice. The female knockout mice are hypertensive and we are determining the levels of isoprostane as a marker of oxidative stress resulting from hypertension. Another study with D. Zeldin was designed to examine the role of CYP2J2 in hypoxia-reoxygenation-induced injury in cultured bovine aortic endothelial cells (BAECs). Transfection of BAECs with the CYP2J2 cDNA resulted in increased CYP2J2 expression and arachidonic acid epoxygenase activity, compared with cells transfected with an irrelevant green fluorescent protein (GFP) cDNA. HR induced significant injury in GFP-transfected BAECs; however, the HR-induced injury was markedly attenuated in CYP2J2-transfected cells (p < 0.01). HR increased cellular 8-iso-prostaglandin F-2 (P < 0.05), and decreased eNOS expression, L-arginine uptake and conversion, and nitrite production (p < 0.01) in GFP-transfected BAECs. CYP2J2 transfection attenuated the HR-induced increase in 8-iso-prostaglandin F-2 alpha (P < 0.05) and decreased the amount of extracellular superoxide detected by cytochrome c reduction under normoxic conditions (p < 0.05) but did not significantly affect HR-induced decreases in eNOS expression, L-arginine uptake and conversion, and nitrite production.